Cells
move for a variety of reasons.Bacteria
swim by beating their flagella in order to exploit newly created
micro-environments and amoeba crawl to gather bacteria to feed on.The cells of vertebrates must move to heal wounds, fend off
invaders and the eukaryotic flagella is used to propel sperm cells
toward eggs.

Cell
type

Speed
(mm/sec)

Bacteria

10

Ciliate

1,000

Amoeba
proteus

3

Neutrophil

0.1

Fibroblast

0.01

Table 1
Speed record of different types of cell locomotion.

Figure
1

The
Prokaryotic Flagellum

Bacteria
invented the wheel! The bacterial flagellum is a helical structure that
drives the cell through the media like a propeller.The structure is rigid and turned by a rotatory motor at the base
where it connects to the bacteria's body.The rotary motor consist of several wheel-like discs one of which
the M-ring (and/or possibly the S-ring) interact with the C-ring and
studs to rotate the whole structure. The rotary motor is very like a
stepping motor! The flagella is composed of a protein called flagellin
which is synthesized in the cell body and transported through the narrow
lumen of the growing flagella itself to polymerise at the tip as it is
about to exit the bacteria!This
system has evolved into a syringe –like mechanism to inject toxins
into the cells of vertebrates during infection (this is called
"type 3 secretion").There
are two main type of prokaryotic flagella, those belonging to gram
positive (one membrane) and gram negative (two membranes) bacteria
(Figure 1).The bacterial
flagellum is driven by a proton motive force resulting from a gradient
of protons.Bacterial
chemotaxis is brought about by alterations in the direction that the
motor rotates in, this in turn is controlled by phosphorylation.

The Eukaryotic FlagellumAlthough
at first sight the flagella of eukaryotes is similar to the flagella of
prokaryotes, our flagella are completely dissimilar in structure,
function and in the genes that encode their components.The principle component of the eukaryotic flagella is the
microtubule, a tubular array of proteins of the tubulin family.Instead of rotating as the prokaryotic flagella does, the
eukaryotic flagella produces contortions in shape that travel around the
structure like a Mexican wave.The
term cilia is generally used to describe small grouped structures less
than 10mm,
and flagella tend to be single structures about 40mm.Our cilia are considered to be a cellular organelle and are
almost certain to be derived from a primitive protist cell in the
distant past.In the human
body they are used in mucus membranes to driven mucus around (out of the
lungs), to drive sperm cells, but bizarrely, in development a single
cilium is responsible for setting up the asymmetry of our internal
organs (heart slightly to the left etc.) rare mutations in the genes
encoding this structure cause situs
inversus.Like many small things, the eukaryotic flagella was first seen by
Anton van Leuwenhoek.The outer doublets are composed of microtubules and the outer
and inner arms are dynein.Dynein
is a motor protein that works with microtubules much like myosin works
on actin so that the whole flagellum is sent into spiral motions as each
set of arms (dynein) walks up the microtubules.

Crawling
locomotion of cells.

Figure
2It
has been suggested that the primary driving force behind the evolution
of the actin cytoskeleton was to permit the equal division of components
at cytokinesis.If this is
so, then a close second must have been the ability to move!The subject of cell locomotion has been a very long one.As far back as primitive microscopes were available scientists
have been studying the so called "Giant amoeba" (Amoeba
proteus & Chaos carolinensis).At 1mm in length, these cells are enormous and so were ideal
subject for the early studies.The fact that these cells moved in "real time"
(3-10mM/sec)
meant that no time lapse photography was necessary either.The large size of these amoebae made it possible to perform
"Micrurgy", such as enucleation and tactile stimulation.It was concluded (Jennings,
1906; Goldacre
1952) from such
tactile studies that prodding the front of the cell caused the cell to
change direction, whereas a gentle prod in the rear made the cell
accelerate transiently (as it would any sensible creature!).
It was found (Goldacre
1953) that the uroid
was contractile and that the cell could move even after the nucleus was
removed with fine glass needle. The finding that the uroid was
contractile was the subject of about a hundred years of controversy as
others (Allen, 1961)
suggested that it was the front end of the cell that was contractile and
active.

Figure
3A

Figure 3B

Observations
on the amoeba revealed that there were two convertible states of
cytoplasm, endoplasm and ectoplasm.Endoplasm (seen at the cell centre, thus the name) was fluid,
while ectoplasm under the cell membrane is gellated and comparatively
static.During active
locomotion, endoplasm flows forwards faster than the speed of the cell.As the fluid endoplasm reaches the "hyaloplasm", a
special optically clear form of ectoplasm, the flow diverted toward the
membrane whereupon the endoplasm gelled to form ectoplasm.Vesicles, crystals, and other visible cytoplasmic inclusions are
seen to become suddenly immobile having previously been seen to vibrate
in Browning motion.

These transformations also take place in other cell types but are
less visible because of the much smaller scale and because they take
place over a much longer time scale.However these transformations are quite clearly visible in small
amoeba such as Acanthamoeba, Dictyostelium and Naegleria,
and also in highly motile human cells such as macrophages and
neutrophils.

The
Frontal Contraction theoryForwarded
by Bob Allen (Allen,
1961) on
the basis of the behaviour of cytoplasm released (accidentally in the
first instance) into media of various formulations.Allen found the liberated cytoplasm had some very peculiar
properties, the cytoplasm was contractile and isolated pools were seen
to writhe producing squirting motions.Allen compared these movements with the reversible gel to sol
conversions that were clearly visible in the living cell.He proposed that as the endoplasm to ectoplasm transformation
took place, the forming ectoplasm contracted pulling the cell forward
(Figure 3A).

The
generalised contraction theoryThe
generalised contraction theory eventually won the day and is more in
favour of the rear contraction hypothesis rather than the frontal
contraction hypothesis.This
hypothesis is only really applicable in its strictest sense to the giant
amoeba as they differ from other cell types in that the cortex is not
attached to the plasma-membrane as it is in smaller amoebae (Dictyostelium,
Acanthamoeba, Naegleria) and vertebrate cells.However, a loose interpretation of the generalised contraction
theory is appropriate for some vertebrate cells at least, as there is a
strong hydraulic component to the locomotion of many cell types.The generalised contraction theory suggests that contraction
occurs as the ectoplasm transforms to endoplasm or in the more modern
parlance, as gel disassembled to sol. Molecular
mechanism have been suggested for this transformation and a body of
experimental evidence supports this (Janson
& Taylor 1993).

Two
types of crawling, lamelapodal spread and hydraulic contraction.
Many cell types in vertebrates adopt a very broad veil-like lamella or
pseudopod at the cell front as they advance forward by crawling.This morphology is best illustrated by the keratinocyte where
most of the projected surface area is occupied by the lamella (figure 4)
(see Lamella expansion).

Figure
4

This cell morphology does not have an obvious hydraulic component.The so called "limax" amoeba (called that because they
look like slugs) are totally hydraulic with no suggestion of
lamellopodal spread.The
neutrophil is intermediate between these two extremes and displays
lamellopodal spread and a hydraulic contraction component. All
cells moving right to left.

The Myosins –
Key motor proteins in Cell Motility and Locomotion.

The
myosins are a group of motor proteins capable of transforming chemical
energy in the form of ATP to movement via the amplification (by levers)
of conformational changes within the ATP hydrolysing head group. Although
there are a huge number of myosin family members we will be discussing
myosin II, this is the two-headed myosin that is the major myosin in
muscle and is responsible for contractile functions (such as
cytokinesis) in the vast majority of non-muscle cells.

Figure
5.Myosin
II. Each
catalytic head group is controlled by two light chains, a regulatory and
an essential light chain.Light
chains are calmodulin-like proteins and wrap around a helical neck
region.The heavy chain
tail regions wrap round each other too.Each myosin II molecule self associates in an anti-parallel
manner regulated by phosphorylation at the C-terminus (P).

Myosin
II performs many functions in cells beside cell locomotion but probably
its most important function is to constrict the waist of the cell and so
allow it to divide.In addition to be regulated by phosphorylation of the light
chains, the heavy chain is phosphorylated at the C-terminus (at the tail
of the myosin molecule).Phosphorylation
here regulates the assembly of the myosin mini-filament, small bipolar
aggregations of myosins that allow the mini-filament to exert a pulling
force on actin filaments.The
assembly of myosin mini-filaments in cells is crucial to myosins
contractile function and is regulated by a large number of different
types of kinases that are in turn activated by a large number of
signalling pathways to regulate their assembly.

B.Myosin light chain kinase phosphorylates the light chain on
threonine 18 and serine 19 inducing a conformational change from a
folded shape, to an extended active shape that is able to form
minifilaments.Phosphorylation at serine 9 by protein kinase C inactivates the
myosin even if it was previously activated by MLCK, furthermore PKC
phosphorylation reduces the rate that the myosin can be phosphorylated
by MLCK.

Figure
6B

Several
myosin heavy chain kinases have been identified (table 4).In Dictyostelium there are three related enzymes MHCK A, B
and C (Liang et al,
2002).The
latter, MHCK C seems to be involved primarily in cytokinesis while forms
A and B were localised with myosin II at the rear cortical region of
moving cells, moreover their distribution here was independent of myosin
II as the same pattern was observed in the absence of myosin II.Dictyostelium also expresses another MHCK that is related
to protein kinase C (Rabin & Ravid,
2002), and like PKC in
vertebrate cells this kinase (MHCK-PKC) inhibits the formation of myosin
filament assembly.MHCK-PKC
is localised at the anterior of the locomoting amoebae rather than the
rear where many of the other kinases are.

Myosin II
filaments are found throughout the cells of the fibroblastic type where
they perform contractile functions unrelated to cell locomotion, but in
cell types where the primary function of myosins is in cell locomotion,
myosin filaments are located at the rear of the cell.This is particularly true for hydraulic cells such as neutrophils
and Dictyostelium.

Myosin
squeezes cytoplasm forward pulls the lamella forward, and rips off
redundant adhesion at the rear.Myosin
II seems to perform three simultaneous functions in cells, the relative
contribution of myosin to these three areas dependent on the particular
locomotory morphology of the cell.In the lamella, myosin II becomes progressively organised in mini-filaments
arranged perpendicular to the direction of locomotion, toward the rear
of the lamella the highest concentration of mini-filaments often
corresponds to the boundary where the lamella meets the cell body.A series of experiments has been reported where cells are settled
onto deformable surfaces onto which small beads have been stuck to act
as position markers.As
cells translocate on these surfaces the pattern of force generation can
be seen.The forces
generated are highly co relatable with the myosin II mini-filament
distribution seen in the various cell types (except in fibroblasts whose
primary function is to contract and not to locomote).It has been possible now to make substrates that are so
deformable that even the very weak forces that Dictyostelium
exerts can be detected and studied (Uchida et al,
2002).The contribution of myosin II to these forces has been assessed
by comparing wild-type Dictyostelium with a strain that lacks
myosin II.The anterior
region produced a pushing force as the cell retracted the posterior and
a pulling forces was detected in the rear.In line with the generalised contraction hypothesis the anterior
pushing force was interpreted as being generated by myosin II squeezing
the cell hydraulically.

The
experimental evidence for the roles of myosin in cell locomotion.Since
the discovery of myosin in non-muscle cells , most hypothesis on the
problem of cell locomotion have been based on the premise that somehow
myosin and actin contractility was involved.It was therefore quite a shock when the single myosin II gene of Dictyostelium
was knocked out and that this did not completely prevent cells from
locomoting (De
Lozanne & Spudich, 1987),
it did however significantly slow them down and prevented the cells
displaying a chemotactic response.The explanation for this is that Dictyostelium only
adheres weakly to the substrate and it has many other myosins through
which it can generate contraction (Dai
et al, 1999).Inhibitory antibodies to myosin II have been microinjected into
adherent Acanthamoeba (Sinard
& Pollard 1989b),
like the situation with Dictyostelium, the removal of myosin II
function slowed but did not stop the cells from moving.Myosin II function has been reduced too in vertebrate cells but
this produced a more radical pattern of disruption (Honer
et al, 1988).The locomotory rate was reduced to almost zero and the cells
seemed incapable of removing rear adhesions resulting in bizarre
morphologies.It seems that
these fibroblasts were more affected that either Dictyostelium or
Acanthamoeba since these cells make much tighter adhesions.Neutrophils cannot be easily microinjected but instead the
contribution of myosin II to locomotion in these cells has been studied
by the treatment of neutrophils with a myosin II inhibitor drug (BDM) (Eddy
et al, 2000),
but the action of this drug is uncertain (see BDM).

Figure
8 Cells locomoting on normal substrates develop adhesions through
which the cell develops force.The behaviour of the beads on the deformable surface (lower
figure).Wrinkles developbetween adhesions where contraction occurs.These wrinkles pull the beads on the latex surface towards the
cell, or in the case of expansion the advancing cell pushes the beads
away.

Figure
9 Myosin II mini-filaments induce contractile tension between
adhesions.As the cell progresses the older focal contacts gather at the
rear of the cell.A combination of focal contact/adhesion disassembly and a build
up of strain results in the release of the adhesion and the elastic
recoil of the material toward the cell body.These recoil evens are often seen to correspond to episodes of
increased protrusion at the leading edge.

Cortical
tube models for cell locomotion

If contraction/solation
of the cortical tube takes place only at the uroid, then the shape of
the cell would be approximately cylindrical.In this most simple of cases a 10% reduction in the volume of the
uroid would result in a 10% increase in volume at the anterior, since
the diameter at each end is equal the width of the cell does not effect
this calculation.However,
most cells are not cylindrical but are variously tapered toward the
uroid.The shape of many
hydraulic cells ectoplasmic cortex is approximately a semi-ellipsoid
(Figure 34a).Sequential
contraction of the cortex by myosin II coupled with solation results in
the formation of sol which can then be recycled to new ectoplasmic
cortex at the front of the cell.Obviously
there is a relationship between contraction and new cortex formation and
this determines the speed of locomotion.If we compute the length of cortical portions created by the same
contraction of cortexes with arbitrary units (Figure 34b) it can be seen
that the width of the cell is not important in determining the speed but
rather it is the length of the cell cortex.This in fact is supported by the literature on amoeba of various
lengths and thicknesses.

Cell
length

Radius100150200

200.6711.3
400.6711.3
800.671 1.3

B.In the above example (Figure 34b) the relationship between the
number of cortical portions produced by each 10% reduction by
contraction of the cortex depends solely on the length and not the cell
width.For a length of 150
units, 1 unit of new cortex is produced by each 10% reduction in volume

Signal
Transduction and Cell Locomotion

As
one might expect, the signal pathways that regulate cell locomotion
overlap to a large degree with those activated during the chemotactic
response.Activated
Rac binds and activates Ca2+ and calmodulin dependent kinase
which can then phosphorylated Myosin II tails causing disassembly.Stimulation of both the PAK1 and PI(3)kinase pathways results in
AKT/PKB phosphorylation and activation through p38MAPK and directly by
PI(3)kinase.AKT/PKB binds
myosin when activates (Tanaka et al,
1999) but the consequence of
this is not yet clear. Activation of MK2 results in the phosphorylation
of myosin light chain and activation of the ATPase.The simultaneous release of the heat shock protein HSP27 may also
affect actin polymerization since this protein (under poorly understood
conditions) binds actin.A
possible negative feed back mechanism may operate through PAK1
phosphorylation of MLCK which inhibits its activity.MLCK is activated by the G-protein Rho and Rho also activated
Rho-kinase which activates myosin by direct phosphorylation of the light
chains.The details of how
these interconnected pathways manage to work together to determine when
and where myosin filaments are formed and when and where they are
activated are not at all clear, but in the neutrophil at least calcium
seems to have a major effect in myosin activation (Eddy
et al, 2000).

Figure 35
Connecting myosin II activation to the major signalling pathways in
neutrophils and Dictyostelium.